Abstract: :
Purpose: 1. To compare the in vitro toxicity of indocyaninegreen (ICG) and infracyanine green (infraCG) to cultured humanRPE cells, and to determine a possible safe dose to utilizefor intraoperative staining of the internal limiting membrane(ILM) during macular hole surgery. 2. To assess the toxicityof two successive ICG exposures of a known safe concentration.Methods: Cultured human RPE cells (ARPE–19, ATCC, Manassas,VA) were grown to confluence and exposed to ICG or infraCG dyeof varying concentrations for exposure times of 3 and 5 minutes.Cells were also exposed to two consecutive 3 minute exposuresof ICG at a concentration of 0.5mg/mL, with double saline washesin between. Following dye exposure, cell viability was measuredand quantified using a well–studied mitochondrial enzymeassay (MTT assay, Sigma Chemicals). All trials were repeateda sufficient number of times to ensure statistical significanceof data.Results: We found that infraCG’s in vitro toxic effectparalleled that of ICG. Using the stock solution infraCG of2.5mg/mL, cell viability measured 48% and 35% following 3 and5 minute exposures respectively. This was similar to that we’destablished for ICG previously, with viabilities of 26% and43% respectively at a concentration of 5mg/mL (stock concentration).Safe concentrations were not seen until a dilution of 0.5mg/mL(i.e. 1/5th the stock concentration) was achieved, with cellviabilities being in excess of 95%. Once again, this resultwas as seen with ICG. When RPE cells were double–stainedwith two consecutive ICG exposures, no measurable decrease incell viability was detected.Conclusions: Our group has previously tested the toxicity ofICG to human RPE cells at various concentrations. In this study,the toxicity of a novel dye, infracyanine green (infraCG), wascompared to that previously determined for ICG. These resultssuggest there is no difference in toxicity of ICG versus infraCGto cultured RPE cells using clinically relevant concentrationsof dye in vitro. To enhance ILM visibility, some surgeons atour center have used two consecutive dye exposures of less thanone minute each using an ICG solution of 0.5mg/mL. In this study,we exposed RPE cells to two consecutive 3 minute exposures ofICG. Cells were washed with balanced salt twice between exposures.Viability of exposed cells showed no significant differencecompared to non–exposed control cells. This would appearto be a safe alternative to using high concentrations of toxicdyes to improve intraoperative ILM detection.